Microtubules are dynamic polymers of the tubulin alpha/beta dimer that provide the tracks for the motor proteins dynein and kinesin and are, therefore, involved in intracellular transport and chromosome separation. Microtubules are the target of anticancer drugs such as the vinca alkaloids and taxol. The assembly and dynamics of microtubules are regulated by GTP in an allosteric two-state model in which tubulin-GTP subunits assemble into microtubules and tubulin-GDP is formed by GTP hydrolysis in the beta-subunit. Our goal is to understand how tubulin works was recently advanced by the determination of its atomic structure. A striking feature of the structure was a loop connecting Helices 7 and 8 (betaH7-8) in the beta-subunit that makes an intradimer contact with a site in the alpha-subunit that binds nonexchangeable and nonhydrolyzable GTP (N-Site) in the alpha- subunit. A similar loop in the alpha-subunit (alphaH7-8) makes an interdimer contact with a site in the beta-subunit that binds exchangeable and hydrolyzable GTP (E-Site). Contact between the alphaH7-8 loop and E-site GTP will accompany tubulin dimer addition to the microtubule end. We will determine the role of the betaH7-8 loop in stabilizing the alpha-beta tubulin intradimer bond, and whether mutations in the loop that are lethal in yeast result from a local effect on the stability of the alpha/beta dimer bond, and/or from long-range effects on the interdimer bond in microtubules. An effect of the intradimer bond will be analyzed from measurements of the Kd for dimer dissociation with mutant tubulins. Effects on the interdimer bond in microtubules will be determined from analysis of microtubule dynamics in living cells. In our studies of the alphaH7-8 loop we will test a hypothesis that this contributes to the GTPase activating activity (GAP) in subunits at microtubule ends that dramatically increases the rate of GTP hydrolysis in subunits when they are incorporated into microtubules. A third goal is determination of the free energy for each of the reactions: Tubulin-GTP-Microtubule reversible reaction Tubulin- GDP-Pi; Tubulin-GDP-Pi reversible reaction Tubulin-GDP+Pi. Measurement of the latter equilibrium will allow us to determine whether with some tubulin isotypes a third form of tubulin, tubulin-GDP-Pi, has a role in regulating microtubule dynamics.